KEYWORDS: Chronic lymphocytic leukemia, Allogeneic hematopoietic cell transplantation, T cell depletion, Alemtuzumab

Transcription

1 The Effect of In Vivo T Cell Depletion with Alemtuzumab on Reduced-Intensity Allogeneic Hematopoietic Cell Transplantation for Chronic Lymphocytic Leukemia Julio Delgado, 1 Srinivas Pillai, 2 Reuben Benjamin, 3 Dolores Caballero, 4 Rodrigo Martino, 1 Amit Nathwani, 3 Richard Lovell, 2 Kirsty Thomson, 3 Jose A. Perez-Simon, 4 Anna Sureda, 1 Panagiotis Kottaridis, 3 Lourdes Vazquez, 4 Karl Peggs, 3 Jorge Sierra, 1 Donald Milligan, 2 Stephen Mackinnon 3 Reduced-intensity conditioning (RIC) allogeneic hematopoietic cell transplantation is increasingly considered for patients with chronic lymphocytic leukemia (CLL). To investigate the impact of in vivo T cell depletion with alemtuzumab on the incidence of graft-versus-host disease (GVHD), nonrelapse mortality (NRM), progression-free survival (PFS), and overall survival (OS), we retrospectively analyzed the outcomes of 62 consecutive CLL patients conditioned with fludarabine and melphalan at 4 institutions. For GVHD prophylaxis, 41 patients (cohort 1) received alemtuzumab and cyclosporin; and 21 patients (cohort 2) received cyclosporin plus methotrexate or mycophenolate. Donors were 50 siblings and 12 unrelated volunteers. Twenty-two (36%) patients received donor lymphocyte infusions (DLI), 20 (49%) from cohort 1 and 2 (10%) from cohort 2 (P 5.002). Grade III-IV acute GVHD (agvhd) was observed in 20% and 38% of patients from cohorts 1 and 2, respectively (P 5.14). Extensive chronic GVHD (cgvhd) was observed in 10% and 48% of patients from cohorts 1 and 2, respectively (P 5.03). There was a trend toward a higher viral infection rate in cohort 1 compared to cohort 2 (68% versus 43%, P 5.062), but the incidence of cytomegalovirus (CMV) reactivation was not significantly different. The 3-year OS, PFS, NRM, and relapse rates were 65%, 39%, 28%, and 32%, respectively, for cohort 1; and 57%, 47%, 34%, and 20%, respectively, for cohort 2 (P 5.629, P 5.361, P 5.735, and P , respectively). In conclusion, both methods of GVHD prophylaxis were equivalent in terms of survival. The administration of alemtuzumab led to reduced cgvhd, possibly improving quality of life. Biol Blood Marrow Transplant 14: (2008) Ó 2008 American Society for Blood and Marrow Transplantation KEYWORDS: Chronic lymphocytic leukemia, Allogeneic hematopoietic cell transplantation, T cell depletion, Alemtuzumab INTRODUCTION B cell chronic lymphocytic leukemia (CLL) is a complex disease characterized by a variable clinical course. A group of patients have an aggressive disease, From the 1 Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 2 Birmingham Heartlands Hospital, Birmingham, United Kingdom; 3 Royal Free & University College Hospitals, London, United Kingdom; and 4 Hospital Clinico Universitario, Salamanca, Spain. Correspondence and reprint requests: Julio Delgado, MD, PhD, Servei d Hematologia Clinica, Hospital de la Santa Creu i Sant Pau, C/Sant Antoni Maria Claret 167, Barcelona, Spain ( jdelgadog@santpau.cat). Received June 17, 2008; accepted September 2, /08/ $34.00/0 doi: /j.bbmt requiring treatment very early after diagnosis, whereas others have an indolent evolution and may not need any kind of therapy for many years [1]. A better understanding of its heterogeneity has emerged from studies defining new prognostic factors such as CD38 expression, ZAP-70 positivity, cytogenetic abnormalities, and immunoglobulin heavy chain variable region (IgV H ) mutations [2]. It is nevertheless recognized that most CLL patients, including around 50% of those with earlystage disease, will eventually die of disease progression or disease-related complications [3]. In addition, up to 30% of patients are younger than 65 years [4], and they almost invariably die of their disease [5]. In some of these patients, sustained responses have been obtained with allogeneic hematopoietic cell transplantation (allo-hct) following conventional 1288

2 Biol Blood Marrow Transplant 14: , 2008 Alemtuzumab in RIC allo-hct for CLL 1289 myeloablative conditioning, particularly in patients with chemosensitive disease at transplantation [6]. Unfortunately, myeloablative transplantation is only suitable for a small proportion of young patients and is hampered by a nonrelapse mortality (NRM) approaching 50% [7]. Reduced-intensity conditioning (RIC) regimens were devised to reduce NRM and extend the use of allo-hct to older patients or those with other medical comorbidities [8]. As such, this procedure is increasingly considered as a therapeutic option for patients with advanced CLL and other lymphoid malignancies [9,10]. There are several RIC regimens, but the majority contain fludarabine (Flu) plus an alkylating agent, usually melphalan (Mel), busulfan (Bu), or cyclophosphamide (Cy) [11]. The Spanish Collaborative Group has relied on the Flu-Mel combination for lymphoid malignancies, using cyclosporin plus methotrexate or mycophenolate mofetil for graft-versus-host disease (GVHD) prophylaxis [12]. The UK Collaborative Group scheme, on the other hand, adds the anti- CD52 monoclonal antibody (mab; alemtuzumab; Campath-1H) to the Flu-Mel combination to deplete recipient and incoming donor T cells, providing sustained engraftment while reducing the incidence of GVHD [13]. This reduced GVHD rate is, however, counterbalanced by a delayed post-hct immune reconstitution, which potentially increases the risk of severe infections and impairs the graft-versus-leukemia (GVL) effect [14]. We report 62 consecutive CLL patients who underwent RIC allo-hct at 4 different institutions: 2 in Spain, and 2 in the United Kingdom. Patients treated in Spain received a standard Flu-Mel conditioning regimen, whereas patients treated in the United Kingdom had alemtuzumab added to Flu and Mel. The aim of the study was to assess the effect of alemtuzumab on GVHD and NRM rates as well as progression-free survival (PFS) and overall survival (OS) in both cohorts. PATIENTS AND METHODS Study Patients We retrospectively collected data from 62 consecutive patients with advanced CLL who underwent RIC allo-hct using a Flu-Mel conditioning regimen from 1999 to The Ethics Committee of each participant center approved all study protocols, and all patients and donors gave written informed consent. Eligibility criteria for transplantation varied across all 4 institutions, but the procedure was generally offered to younger patients with chemorefractory disease, early relapse, and/or poor risk cytogenetic abnormalities, who had a suitable donor. Eighteen patients from the UK were included in the Campath De-escalation Protocol, which analyzed the effect of alemtuzumab dose reduction on the incidence of GVHD and infectious complications. Six patients treated in Spain were recruited for the TIRCAMPATH-Alo-2002 clinical trial, which randomly allocated patients with lymphoproliferative disorders undergoing RIC allo-hct to Flu-Mel or Flu-Mel-alemtuzumab conditioning regimen. Fluorescein in situ hybridization (FISH) cytogenetic studies and analysis of the mutation status of the immunoglobulin gene were performed using conventional methods as previously described [15]. Patients were excluded if they were older than 70 years, the left ventricular ejection fraction was \40%, creatinine clearance was \30 ml/min per 1.73 m 2, or serum bilirubin level or liver transaminases were.3 times the upper limit. Conditioning Regimen, GVHD Prophylaxis, and Stem Cell Source The conditioning regimen comprised Flu 30 mg/ m 2 daily from days 27 to23 (total dose, 150 mg/ m 2 ), and Mel 140 mg/m 2 on day 22. For GVHD prophylaxis, 40 patients received alemtuzumab ( mg) and cyclosporine (cohort 1); and 21 patients received cyclosporine plus MTX or mycophenolate mofetil (MMF; cohort 2). The standard alemtuzumab dose for cohort 1 was 20 mg/day from days 28 to 24 (total dose, 100 mg). However, 18 patients received a total dose of 60 mg (8 patients), 40 mg (3 patients), 30 mg (2 patients), and 20 mg (5 patients) alemtuzumab on days 22 and 21 as part of the Campath De-escalation Protocol (see Study Patients section). Cyclosporin was administered at 3 mg/kg daily starting on day 21. Patients in cohort 2 whose donor was an HLA-identical sibling were also given MTX 15 mg/m 2 on day 11 and 10 mg/m 2 on days 13 and 16 in addition to cyclosporin. Patients in cohort 2 receiving an allograft from an unrelated donor were given MMF 15 mg/kg/day twice daily from day 11 instead of MTX. Two patients in cohort 2 allografted from single-locus mismatched unrelated donors were given antithymocyte globulin (ATG; 2 mg/kg/day from days 24 to21) in addition to cyclosporin and MMF. In the absence of GVHD, cyclosporin 6 MMF were tapered from 3 months after transplantation. Acute and chronic GVHD (agvhd, cgvhd) were graded according to standard criteria [16,17]. In particular, extensive cgvhd was defined as either (1) generalized skin involvement; (2) localized skin involvement, and/or liver dysfunction plus involvement of 1 other target organ; or (3) involvement of at least 2 target organs. Steroid-refractory GVHD was defined as no response to corticosteroids (eg prednisone 2 mg/ kg or equivalent) administered for at least 5 consecutive days or progression after 48 hours of therapy.

3 1290 J. Delgado et al. Biol Blood Marrow Transplant 14: , 2008 On day 0, patients received peripheral blood stem cells (PBSC) from HLA-matched siblings (n 5 50) or unrelated donors (n 5 12). Patients and their donors were matched for HLA-A, -B, -C, -DRB1, and -DQB1 by intermediate- or high-resolution DNA-typing techniques, as appropriate. Six unrelated donor/recipient pairs had single-allele mismatches. Chimerism, Donor Lymphocyte Infusions (DLI), and Supportive Care Whole blood and lineage-specific chimerism (in T cell and myelogenous lineages) were assessed by means of polymerase chain reaction (PCR) analysis of informative minisatellite regions or FISH for X and Y chromosomes in the event of sex-mismatched transplantations. Mixed chimerism was defined as the presence of more than 5% and\95% CD3 1 cells from the donor. Patients who had mixed chimerism or residual disease 6 months after transplantation were eligible to receive donor lymphocyte infusions (DLI) if there was no evidence of active GVHD. Escalating doses of CD3 1 lymphocytes were administered starting at a dose of T cells/kg. Increasing doses were administered at 3-month intervals (3 10 6,110 7,3 10 7, and T cells/kg) in the absence of GVHD if mixed chimerism or residual disease persisted. Infection prophylaxis varied across all participating centers but included antifungals (fluconazole, itraconazole, or voriconazole), aciclovir and cotrimoxazole, or pentamidine. Patients with persistent fever during neutropenia were treated with broad-spectrum antibiotics. Additional agents (glycopeptides, aminoglycosides, antifungals, or antivirals) were added as clinically indicated. Patients were not routinely screened for Epstein-Barr virus (EBV) reactivation. Patients at risk for cytomegalovirus (CMV) reactivation were monitored weekly by antigenemia or quantitative PCR. When the CMV PCR was positive, the test was repeated and preemptive treatment with ganciclovir or its alternatives (valganciclovir, foscarnet) was started if the result was confirmed. Definitions and Statistical Analysis Disease response was evaluated using National Cancer Institute Working Group (NCI-WG) criteria [18]. Flu-refractory CLL was defined as disease that failed to respond to Flu-based therapy or progressive disease within 6 months of response to a Flu-based regimen. Where possible, patients in complete remission (CR) underwent multiparametric flow cytometry analysis on bone marrow samples every 3-6 months to detect minimal residual disease (MRD). MRD determination assays varied across all institutions, but positive MRD was defined as.1% clonal CD5/ CD19 cells in the bone marrow. Time-to-event outcomes with competing risks (ie, NRM and relapse rates) were estimated by cumulative-incidence curves. Comparison of cumulativeincidence curves was performed by the Lunn-McNeil approach in which Cox regression analysis is applied to competing risks [19]. OS and PFS were estimated by the Kaplan-Meier method and compared using the log-rank test. PFS estimates do not include responses to DLI. Several factors were analyzed for their association with OS, PFS, and NRM, including age (50 years and younger versus.50 years), FISH abnormalities (unfavorable versus favorable), IgV H mutation status (mutated versus unmutated), donor type (sibling versus unrelated), status at transplantation (progressive disease [PD] versus CR 1 partial remission [PR]), previous autologous transplantation, number of previous chemotherapy regimens (\3 versus 3 or more), alemtuzumab in the conditioning regimen, and Flu refractoriness. Subsequently, multivariate analysis according to the Cox proportional hazards regression model was used to explore the independent effect of variables that showed a significant influence on OS or PFS by univariate analysis. In all statistical calculations, a value of P \.05 were considered significant. RESULTS Patient Characteristics Patients baseline characteristics were not significantly different between cohorts, and are depicted in Table 1. Median age at transplantation was 53 years (range: 34-64), and 73% of patients were male. FISH prognostic information was available in 38 patients (61%); using the hierarchical model proposed by Döhner et al. [20], 12 of them (32%) had 17p deletion, 13 (34%) had 11q deletion, 4 (11%) had trisomy 12, and 9 (24%) did not have any detectable aberration. IgV H mutation status data was available in 35 patients (57%). Using the conventional 2% cutoff level, 28 patients (80%) were classified as unmutated CLL, while the remaining 7 patients had mutated CLL. CD38 or ZAP-70 expression data were only available in a small proportion of patients and therefore not considered. Two patients from cohort 1 experienced high-grade (Richter) transformation before transplantation. Transplantation took place at a median of 55 months after the initial CLL diagnosis (range: months). Before the procedure, patients received a median of 3 chemotherapy regimens (range: 1-6). These regimens varied across all 4 institutions, but 87% of patients received Flu, 19% received alemtuzumab, and 29% received rituximab before allo-hct. Of 54 patients who received Flu prior to transplantation, 21 (39%) were considered refractory according to

4 Biol Blood Marrow Transplant 14: , 2008 Alemtuzumab in RIC allo-hct for CLL 1291 Table 1. Patients Baseline Characteristics Cohort 1: Alemtuzumab + CSP (n 5 41) Cohort 2: CSP + MTX or MMF (n 5 21) P Value (Fisher s Exact or Mann-Whitney s Test) Age in years (median, range) 52 (37-64) 54 (34-64).451 Sex (% male/female) 73/27 71/ Unmutated IgV H genes (n, %) 15 (83%) 13 (77%).691 Cytogenetics by FISH p deletion + 11q deletion (n, %) 14 (74%) 11 (58%) Number of previous therapy lines (median, range) 3 (1-4) 3 (1-6).620 Previous fludarabine (n, %) 37 (90%) 17 (81%).426 Previous alemtuzumab (n, %) 7 (17%) 5 (24%).520 Previous rituximab (n, %) 15 (37%) 3 (14%).082 Prior autologous HCT (n, %) 7 (17%) 2 (10%).705 Fludarabine refractoriness (n, %) 16 (43%) 5 (29%).383 Months to first therapy (median, range) 1.5 (0-59) 4 (0-59).429 Months to allogeneic HCT (median, range) 56 (5-105) 47 (11-132).817 Disease status at HCT.054 Complete or partial remission (n, %) 35 (85%) 13 (62%) Progressive disease (n, %) 6 (15%) 8 (38%) Donor.639 Matched related (n, %) 32 (78%) 18 (86%) Matched unrelated (n, %) 5 (12%) 1 (5%) Mismatched unrelated (n, %) 4 (10%) 2 (9%) CMV positive donor or recipient (n, %) 34 (83%) 19 (91%).705 Follow-up from HCT (median, range) 38 (6-103) 59 (25-102).066 CSP indicates cyclosporin; MTX, methotrexate; MMF, mycophenolate mofetil; IgV H, variable region of the immunoglobulin heavy-chain gene; FISH, fluorescein in situ hybridization; HCT, hematopoietic cell transplantation; CMV, cytomegalovirus. National Cancer Institute (NCI) criteria [18]. Also, 9 patients (15%) had previously failed an autologous HCT. At the time of transplantation, 6 patients (10%) were in complete remission, 42 (68%) were in partial remission, and 14 (23%) had progressive CLL. There was a trend toward a significantly higher proportion of patients with progressive disease in cohort 2 (38%) compared to cohort 1 (15%) (P 5.054, Table 1). Engraftment, Donor Chimerism, and GVDH The graft source was PBSC for all patients. All but 1 patient had stable engraftment. The median interval to neutrophil recovery ( /L) was 14 days (range: 10-36), and the median time to platelet recovery ( /L) was 12 days (range: 8-40). The incidence of mixed chimerism at 6 months posttransplantation was significantly higher in patients from cohort 1 (43%) compared to patients from cohort 2 (11%) (P 5.03, Fisher s exact test). However, the majority of these patients with mixed chimerism received DLI and the global incidence of secondary graft failure was 11% (95% confidence interval [CI], 3%-19%), with no significant differences between cohorts (see Table 2). Grade II-IV agvhd, including post-dli GVHD, was diagnosed in 15 patients (37%) from cohort 1 and 12 patients (57%) from cohort 2 (P 5.18). Severe (grade III-IV) agvhd was observed in 8 (20%) and 8 (38%) patients from cohorts 1 and 2, respectively (P 5.14). Four patients (10%) from cohort 1 developed steroid-refractory GVHD compared to 7 patients (33%) from cohort 2 (P 5.03). cgvhd was documented in 12 (29%) and 13 (68%) patients from cohorts 1 and 2, respectively (P 5.016). Extensive cgvhd requiring systemic therapy was observed in 4 patients (10%) from cohort 1 and 10 patients (48%) from cohort 2 (P 5.03). As a result, GVHD-related crude mortality was 10% (1%-19%) for patients in cohort 1 and 33% (12%- 54%) for patients in cohort 2 (P 5.034). Of all 10 patients with extensive cgvhd that are still alive, 6 are on immunosuppressive agents, 2 from cohort 1, and 4 from cohort 2. Infections Severe infections were common in both cohorts, with no significant differences between them (73% versus 67%, P 5.768, Table 3). Eleven patients (18%) died of infectious complications, 7 of them GVHD related. These infectious deaths were equally distributed between cohorts (20% versus 14%, P 5.735), and comprised septicemia (4 patients), EBV-related posttransplantation lymphoproliferative disease (3 patients), bacterial pneumonia (1 patient), bacterial meningitis (1 patient), pulmonary aspergillosis (1 patient), and respiratory syncytial virus (RSV) pneumonia (1 patient). Globally, there was a trend toward a higher viral infection rate in cohort 1 compared to cohort 2 (68% versus 43%, P 5.062). These were caused by herpes simplex (7 patients), EBV (6 patients, including 3 posttransplantation lymphoproliferative disorder [PTLD] cases), adenovirus (5 patients), RSV (4 patients), influenzae (3 patients), varicella zoster (2 patients), metapneumovirus (1 patient), and parainfluenzae

5 1292 J. Delgado et al. Biol Blood Marrow Transplant 14: , 2008 Table 2. Results after Reduced Intensity Allogeneic Hematopoietic Cell Transplantation Cohort 1: Alemtuzumab + CSP (n 5 41) Cohort 2: CSP + MTX or MMF (n 5 21) P Value (Fisher s Exact or Mann-Whitney s Test) Primary engraftment failure (n, %) 1 (2%) 0 (0%) 1.0 Time to neutrophil recovery [> /L] in days (median, range) 14 (10-27) 14 (13-36).283 Time to platelet recovery [> /L] in days (median, range) 12 (8-40) 13 (10-39).132 Chimerism at 6 months (n, %).030 Mixed 16 (43%) 2 (11%) Full donor 21 (57%) 16 (89%) Secondary engraftment failure (n, %) 5 (12%) 2 (9%) 1.0 NRM at 3 months (n, %) 0 (0%) 3 (14%).035 NRM at 1 year (n, %) 8 (20%) 5 (24%).748 NRM at 3 years (n, %) 11 (27%) 7 (33%).768 Acute GVHD Grace II-IV (n, %) 15 (37%) 12 (57%).117 Grade III-IV (n, %) 8 (20%) 8 (38%).135 Total (n, %) 21 (51%) 14 (67%).288 Chronic GVHD Extensive (n, %) 4 (10%) 10 (48%).003 Total (n, %) 12 (29%) 13 (62%).016 Steroid-refractory GVHD (n, %) 4 (10%) 7 (33%).034 Cytomegalovirus reactivation (n, %) 21 (62%) 8 (42%).250 Use of DLI (n, %) 20 (49%) 2 (10%).002 CSP indicates cyclosporin; MTX, methotrexate; MMF, mycophenolate mofetil; NRM, nonrelapse mortality; GVHD, graft-versus-host disease; DLI, donor lymphocyte infusions. (1 patient). Forty-nine patients (79%) were at risk of CMV reactivation: 32 patients in cohort 1 and 17 patients in cohort 2. The incidence of CMV reactivation was not significantly different between cohorts (66% versus 47%, P 5.237). There was a trend toward a higher EBV reactivation rate, including 3 cases of PTLD, in patients from cohort 1 (15% versus 0%, P 5.088). Regarding fungal and bacterial infections, they were equally distributed between cohorts (P 5 Table 3. Severe Infections According to Conditioning Regimen Cohort 1: Alemtuzumab + CSP (n 5 41) Cohort 2: CSP + MTX or MMF (n 5 21) Viral Cytomegalovirus 21/32 (66%) 8/17 (47%) reactivation Epstein-Barr 6 (15%) 0 (0%) (reactivation or PTLD) Respiratory syncytial 4 (10%) 0 (0%) Adenovirus 5 (12%) 0 (0%) Herpes simplex 5 (12%) 2 (10%) Varicella zoster 1 (2%) 1 (5%) Influenzae 2 (5%) 1 (5%) Metapneumovirus 1 (2%) 0 (0%) Parainfluenzae 1 (2%) 0 (0%) Bacterial Pseudomonas aeruginosa 2 (5%) 4 (19%) Mycobacterium spp. 2 (5%) 0 (0%) Nocardia asteroides 1 (2%) 0 (0%) Campylobacter jejuni 0 (0%) 2 (10%) Salmonella enterica 0 (0%) 1 (5%) Escherichia coli 0 (0%) 2 (10%) Klebsiella pneumoniae 0 (0%) 1 (5%) Fungal Aspergillus spp. 2 (5%) 2 (10%) Pneumocystis jirovecci 1 (2%) 1 (5%) CSP indicates cyclosporin; MTX, methotrexate; MMF, mycophenolate mofetil; PTLD, posttransplant lymphoproliferative disorder..38 and P 5 1.0, respectively). The most commonly isolated pathogens were Aspergillus spp. (4 patients) and Pseudomonas aeruginosa (6 patients), and there were 2 cases of Pneumocystis jirovecci pneumonia. Disease Response, DLI, and Survival The overall complete response rate among 56 patients with measurable disease at the time of transplantation was 75% (64%-86%), whereas 18% (8%- 28%) had stable disease. Four patients (7%) were never restaged after transplantation because of early death (3 patients) or unknown reasons (1 patient). The complete response rate was 70% (55%-85%) for cohort 1 and 84% (68%-100%) for cohort 2 (P 5.514). Twenty-two patients (36%) received escalated DLI: 20 patients (49%) from cohort 1 and 2 patients (10%) from cohort 2 (P 5.002). Reasons for DLI were mixed chimerism in 9 patients, MRD in 5 patients, and residual or progressive CLL in 8 patients. Of 9 patients with mixed chimerism, all but 1 achieved full-donor chimerism, but 2 additional patients have subsequently relapsed. Regarding patients with minimal residual disease or clinical disease relapse, 6 patients responded to DLI, but only 2 are currently free of disease. With a median follow-up of 46 months (range: 6-103), 24 patients (39%) have died, 5 of progressive disease, and 19 of transplant-related complications. Cumulative incidences of NRM at 3 years were 28% (17%-47%) for cohort 1 and 34% (18%-62%) for cohort 2, respectively (P 5.735). Conversely, the 3-year relapse rates were 32% (20%-52%) for cohort 1 and 20% (8%-47%) for cohort 2 (P 5.112) (Figure 1). Kaplan-Meier estimates of OS and PFS are shown in

6 Biol Blood Marrow Transplant 14: , 2008 Alemtuzumab in RIC allo-hct for CLL 1293 Figure 1. NRM (A) and relapse rate (B) according to conditioning regimen (with alemtuzumab, gray line; without alemtuzumab, black line; P and.112, respectively). Both outcomes were considered competing risks for the purpose of this analysis. Figure 2. Estimated OS and PFS rates at 3 years were 65% (49%-81%) and 39% (23%-55%), respectively, for cohort 1 and 57% (35%-79%) and 47% (25%- 69%), respectively, for cohort 2. The log-statistic showed no statistical differences (P for OS and P for PFS). The only variable with a significant impact on OS was Flu refractoriness before transplantation (P 5.009, log-rank test). DISCUSSION The hematology community has recently witnessed the appearance of several new agents, including rituximab combinations [21], alemtuzumab [22], ofatumumab [23], flavopiridol [24], or lenalidomide [25] for young patients with relapsed or refractory CLL. It is, however, uncertain at this time whether these promising drugs will lead to prolonged survival or not. In the past decade, multiple reports from both sides of the Atlantic have suggested that RIC allo-hct may be a potentially curative strategy for these patients [12,14,26-30]. The aim of this approach is to deliver the advantages of the GVL effect without the NRM associated with conventional allo-hct [11]. Recently, the European Group of Blood and Marrow Transplantation (EBMT) has recently published a set of guidelines suggesting situations where allo-hct might be considered a therapeutic option for CLL patients. Their conclusion was that allo- HCT was reasonable for younger CLL patients refractory to Flu, failing autologous HCT, or with p53 abnormalities requiring treatment [10]. Once a patient is considered a suitable candidate for allo-hct, the next important decision is the type of conditioning regimen to use. The EBMT Consensus Group suggested that RIC allo-hct is effective for poor-risk CLL, regardless of its comparison to conventional myeloablative allo-hct [10]. In addition, a recent retrospective comparison from the Seattle Group revealed that CLL and lymphoma patients with comorbidities, whether young or old, Figure 2. OS (A) and PFS (B) according to conditioning regimen (with alemtuzumab, gray line; without alemtuzumab, black line; P and.361, respectively).

7 1294 J. Delgado et al. Biol Blood Marrow Transplant 14: , 2008 had better survival after RIC compared to conventional allo-hct. The same study also showed that the conditioning regimen only played a minor role in terms of disease control [30]. There is, however, considerable controversy regarding the potential benefit of T cell depletion (TCD), either with alemtuzumab or other methods. For instance, a recent multicenter randomized trial assessed the impact of TCD by physical and immunologic methods on unrelated donor bone marrow transplantation. Interestingly, the 3-year disease-free survival (DFS) was similar for both TCD and non-tcd allografts, whereas agvhd rates were significantly lower for patients receiving TCD grafts. Relapse rates were not adversely affected by TCD, with the only exception of patients with chronic myelogenous leukemia (CML). The conclusion of this study was that TCD did not enhance patient survival despite reducing GVHD rates and early toxicity [31]. We therefore decided to compare retrospectively the results obtained in patients with poor-risk CLL undergoing RIC allo-hct with or without alemtuzumab. One of the strengths of this study is the reduced number of participant centers, which was specifically sought after to avoid the center effect. The center effect has long been recognized as a very important variable for patients undergoing allo-hct for acute myelogenous leukemia (AML) [32]. In CLL, we had previously observed a potential center effect in patients undergoing RIC allo-hct [14], and therefore decided to select 2 transplant centers from Spain and 2 from the UK. All 4 institutions have performed a significant number of procedures in this group of patients and have similar NRM rates. In addition, all patients were conditioned with a Flu plus Mel regimen, with or without alemtuzumab. In concordance with a previous similar study including all types of lymphoid malignancies [33], this analysis confirms that alemtuzumab significantly reduces the cgvhd and steroid-refractory GVHD rates, as well as the GVHD-related mortality. There was also a trend toward a reduction in grade II-IV agvhd. The low GVHD rates detected in cohort 1 are consistent with previous reports using alemtuzumab [14]. Also, the cgvhd rates obtained in cohort 2 is very similar to those observed in other centers [28], and almost identical to the results obtained by the Spanish Cooperative Group using the same conditioning regimen in patients with AML [34]. These facts make us believe that this difference in GVHD rates observed in our study is genuine. This reduced GVHD rate was, however, partially offset by a trend toward an increased relapse rate for patients receiving alemtuzumab, which did not translate into a significantly worse PFS or OS. This contrasts with the outcomes achieved with RIC allo-hct in multiple myeloma (MM) or CML, where the addition of alemtuzumab or any T cell-depleting agent is potentially detrimental [31,33,35,36]. It is, however, remarkable that no patient from cohort 2 has relapsed later than 3 years after HCT, whereas a number of late relapses have been observed in cohort 1. Perhaps, a similar study with more patients would have shown a significantly different PFS between cohorts [33]. This analysis has also shown that CLL may be susceptible to GVL effect [37]. The incidence of mixed chimerism after transplantation was significantly higher in patients from cohort 1. DLI were almost exclusively given to patients from cohort 1 and were very effective for transforming mixed chimerism into complete donor chimerism. DLI were nevertheless less effective for relapsed or progressive disease. Even though 6 of 13 patients (46%) who received escalated DLI for MRD or clinical progression initially responded to the measure, only 3 patients (23%) are currently free of CLL. These results confirm previous reports showing response rates around 15% for CLL patients receiving DLI for relapsed disease after HCT [28,38-40]. In contrast, the MD Anderson group has recently reported response rates to DLI in excess of 50% for patients with progressive CLL following RIC allo-hct, but interpretation of their studies is complicated by the concomitant administration of rituximab [41]. Infections were frequent, and contributed to a significant proportion of deaths and morbidity. However, patients from cohort 1 did not experience increased CMV reactivation rates compared to patients from cohort 2, in contrast to previous studies [33]. We believe this is because of the fact that poor-risk CLL patients are intrinsically prone to infections regardless of the conditioning regimen received. This susceptibility to infections is disease and therapy related, and is secondary to multiple factors, including hypogammaglobulinemia, defective T and natural killer (NK) cell function, neutropenia, and deficient complement activity [42]. Indeed, it is widely acknowledged that infections account for up to 50% of all CLL-related deaths [15,43]. Furthermore, alemtuzumab depletes incoming T cells and delays the immune reconstitution after allo-hct. However, we hypothesize that the detrimental effect of alemtuzumab in cohort 1 was counterbalanced by the greater risk of extensive cgvhd observed in cohort 2. This increased GVHD rate led to the administration of systemic corticosteroids or very potent immunosuppressive agents, such as inolimomab or daclizumab, which may have compromised their immune reconstitution even further. A major concern about the use of TCD in allo- HCT is the increased incidence of EBV-related PTLD. Several studies have concluded that the risk of PTLD depends greatly on the method of TCD used, being considerably higher for specific T cell

8 Biol Blood Marrow Transplant 14: , 2008 Alemtuzumab in RIC allo-hct for CLL 1295 antibodies (eg, thymoglobulin) compared to T and B cell antibodies (eg, alemtuzumab) [44]. In this study, 3 patients from cohort 1 developed PTLD, which was also their cause of death. This prompted a strict virologic surveillance by means of quantitative PCR for all patients receiving alemtuzumab as part of their conditioning regimen, and 3 further patients were noted to have EBV reactivation. Preemptive administration of rituximab in these 3 patients was sufficient to prevent the development of PTLD. As previously reported [14], secondary graft failure was observed in a significant proportion of patients from both cohorts (12% versus 9%, P 5 1.0). We speculate that this phenomenon is disease related, as there is some evidence that sustained allogeneic engraftment is mediated by host dendritic cells, which are seriously defective in CLL patients [45]. As a matter of fact, a recent report on unrelated transplantation for CLL using myeloablative conditioning regimens revealed an even higher incidence (18%) of graft failure [46]. Flu refractoriness confirmed its significant impact on OS as previously observed [14]. Several other groups have analyzed the effect of response to prior chemotherapy, particularly Flu, as a major prognostic factor for patients undergoing allo-hct. The MD Anderson and other groups have recently published updated results showing that chemorefractoriness at the time of transplantation is a very important predictive factor in terms or disease relapse and survival [29,41]. In our series, Flu refractory patients had a median OS of 17 months following allo-hct, which is still insufficient and marginally better than historic results obtained with conventional chemotherapy (10 months) or alemtuzumab (16 months) as salvage therapy [47,48]. Alternative therapies or transplantation regimens are clearly needed for these patients. For the time being, we would favor the use of alemtuzumab in patients receiving their grafts from unrelated donors, particularly if they are mismatched, unless there is a very high relapse risk (eg, patients with chemorefractory disease at transplantation). For the remaining patients the jury is still out, and we would base our recommendation on physician s preference, the availability of cells for DLI, molecular techniques for CMV and EBV detection, and so forth. We would like to acknowledge that the conclusions drawn from this retrospective analysis are less compelling than those from randomized prospective studies. However, in the absence of prospective randomized trials requiring multicenter collaboration, retrospective analyses may provide adequate data to continue improving our clinical practice. Another weakness of the study is the diverse alemtuzumab dose received by patients from cohort 1, as many patients were recruited in the Campath De-escalation Protocol. This trial has recently completed its recruitment, and will try to establish the alemtuzumab dose that provides the best GVHD protection with the lowest relapse rate. Unfortunately, low patient numbers did not allow us to evaluate the impact of each alemtuzumab dose on NRM, GVHD rates, and survival, and this will be the subject of future publications including the whole cohort. Finally, it could be argued that the statistical power of the study was suboptimal as cohort 1 included only 21 patients. After much consideration, we decided that it was better to have a small cohort of patients receiving the same conditioning regimen at 2 experienced institutions rather than a more heterogenous cohort receiving diverse regimens at many different institutions. 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